Modeling the LPS-induced effects on transcription factor activation and gene expression in murine macrophages.

Macrophages within the liver are of particular importance for a functional defense against bacterial infection. They exhibit a complex response to lipopolysaccharide and secrete a variety of pro-inflammatory cytokines and chemokines that both coordinate the immune response and regulate activity of the macrophages, themselves. In this context, the dynamic of pathway activation and gene expression is important for a better understanding of the role of activated macrophages in healthy and diseased states. Therefore, we present a representative model of LPS-induced macrophage activation that covers the principle regulatory motifs. Based on that, we propose a simplified model with a reduced number of states and parameters that allows estimation of transcription factor activity from gene expression data and can be easily extended to describe the full spectrum of gene regulation in LPS-activated macrophages.

[Hepatic cholestasis resulting from a long-standing enterothorax: a case report]. / Langjähriger Enterothorax als ungewöhnliche Ursache einer Cholestase: ein Fallbericht.

BACKGROUND: Due to the lack of symptoms an enterothorax frequently remains undetected in adults. Most symptomatic patients complain about bowel obstruction and a surgical repair of the diaphragmatic defect, particularly with a mesh, is mandatory. METHODS: This report presents the case of a 72-year-old female patient with a history of an upside-down stomach presenting with a painless jaundice and signs of liver cirrhosis. CLINICAL COURSE: The preoperative work-up revealed an enterothorax with compression of the main bile duct. Explorative laparotomy showed a liver cirrhosis with distinct intrahepatic cholestasis, a hydropic gallbladder and confirmed a right-sided diaphragmatic defect with an enterothorax. After reposition of the intestine, a cholecystectomy, bile duct revision and the closure of the diaphragmatic defect using a mesh were performed. CONCLUSION: Diaphragmatic defects are the basis for the formation of an enterothorax which may be associated with a complicated clinical course. Therefore, in cases of coincidental diagnosis, even in asymptomatic patients, surgical repair should be performed in order to prevent serious complications as presented in this case.

Multiple liver abscesses with isolation of Streptococcus intermedius related to a pyogenic dental infection in an immuno-competent patient.

INTRODUCTION: Streptococcus intermedius - a member of the Streptococcus anginosus group - is part of the normal microbial flora of the oral cavity. Despite being regarded as a harmless apathogenic commensal, Streptococcus intermedius has been described to cause abscesses in various locations of the body. CASE PRESENTATION: We report the clinical case and course of treatment of a 18-year-old male patient presenting with multiple hepatic abscesses associated with an untreated pyogenic dental infection. CONCLUSION: Streptococcus intermedius can cause liver abscesses emerging from dental infectious foci even in previously healthy patients without underlying innate or aquired immunodeficiency. The case illustrates the potential danger and underestimated risk associated with untreated dental infections.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor alpha mediated liver disease.

BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Differential effects of vasodilatory prostaglandins on focal adhesions, cytoskeletal architecture, and migration in human aortic smooth muscle cells.

OBJECTIVE: Cyclooxygenases 1 and 2 are expressed in atherosclerotic arteries, and local generation of prostacyclin and prostaglandin E2 (PGE2) occurs. However, the role of cyclooxygenases and individual prostaglandins during plaque progression is currently uncertain. The present study characterizes the effect of vasodilatory prostaglandins on morphology, focal adhesion (FA) function, and migration in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: The stable prostacyclin analog iloprost transiently induced: (1) disassembly of FA and stress fibers, (2) partial retraction and rounding of SMCs, (3) hypophosphorylation of FA kinase (FAK) and paxillin, and (4) inhibition of platelet-derived growth factor-BB-induced migration. Inhibition of FAK phosphorylation and morphological changes were mimicked by forskolin, inhibited by H89, and prevented by the protein tyrosine phosphatase inhibitor vanadate and by calpeptin. PGE2 was by far less efficient with respect to all parameters investigated. This difference correlated with the respective cAMP induction in response to iloprost and PGE2. CONCLUSIONS: Inhibition of FAK phosphorylation and FA function is a new target of vasodilatory prostaglandins, which might be causally involved in the antimigratory effects of prostaglandins. Importantly, prostacyclin analogs and PGE2 differ dramatically with respect to dephosphorylation of FAK and inhibition of migration, which might be of relevance for their respective functions in atherosclerosis.

The MKK6/p38 mitogen-activated protein kinase pathway is capable of inducing SOCS3 gene expression and inhibits IL-6-induced transcription.

In this study we show that activation of p38MAPK by IL-6 acts as an inhibitory signal on IL-6-mediated activation of STAT and the alpha2-macroglobulin promoter. We analyzed the role of MKK6/p38MAPK for IL-6 signal transduction and transcriptional activation of the suppressor of cytokine signaling (SOCS) 3 promoter. Pretreatment of cells with the p38MAPK-specific inhibitor SB202190 downregulates the induction of SOCS3-mRNA expression by IL-6. Accordingly, overexpression of a constitutively active MKK6 in HepG2 cells enhanced basal activity or IL-6-induced transcriptional activation of a SOCS3 promoter reporter construct, whereas overexpression of a dominant negative mutant of MKK6 downregulated the IL-6-mediated activation of the SOCS3 promoter. These data indicate that p38MAPK-activation is crucial for IL-6-induced SOCS3 expression and downregulation of IL-6-mediated gene induction.

The inhibitory effect of IL-1 beta on IL-6-induced alpha 2-macroglobulin expression is due to activation of NF-kappa B.

The cross-talk between the signal transduction of simultaneous acting cytokines largely determines the final impact of cytokines on their target genes. Both NF-kappaB and STAT3 are transcription factors well known to be activated by many stimuli and to mediate transcriptional activation by binding to specific enhancer sequences. In this study, it is analyzed how IL-1beta inhibits IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. It is shown that IL-1beta prevents STAT3 binding to the two STAT3-responsive sites within the alpha(2)-macroglobulin promoter by association of IL-1beta-activated NF-kappaB to this region. The observation that inhibition of IL-6-induced transcriptional activation of this promoter by IL-1beta is reversed by cotransfection with I-kappaBalpha provides evidence that NF-kappaB activation by IL-1beta is responsible for inhibition of IL-6-mediated trans activation of the alpha(2)-macroglobulin gene. Accordingly, cotransfection of the NF-kappaB subunits p50 or p65 themselves inhibited activation of the alpha(2)-macroglobulin promoter by IL-6. Introduction of point mutations in each of the two NF-kappaB sites overlapping the two STAT3 binding sites within the alpha(2)-macroglobulin promoter provides evidence that each of these two sites counteracts transcriptional activation via STAT3. Most interestingly, at least one functional NF-kappaB consensus site is essential for the IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. Additional data are provided indicating that the activation of NF-kappaB by IL-1beta is also responsible for the inhibition of other IL-6-inducible genes, such as the alpha(1)-antichymotrypsin gene as well as the suppressor of cytokine signaling 3 gene, suggesting a more general relevance of this mechanism for transcriptional regulation.

Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells.

BACKGROUND AND AIMS: Hepatic stellate cell (HSC) transformation and proliferation play an important role in liver fibrogenesis, and HSC apoptosis may be involved in the termination of this response. METHODS: Expression of the peripheral benzodiazepine receptor (PBR) and PBR-ligand-induced apoptosis were studied in cultured rat liver HSC. RESULTS: Transformation of HSC led to a transient expression of PBR at the messenger RNA and protein level, which was maximal after about 3 and 7 days of culture, respectively, and declined thereafter. Immunoreactive PBR showed a punctate staining and colocalized with mitochondrial manganese-dependent superoxide dismutase and adenine nucleotide translocator 1. The selective PBR ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and 4' chlorodiazepam (Ro5-4864), but not the centrally acting benzodiazepine ligand clonazepam, induced dose-dependent apoptosis in HSC. The apoptotic potency of PK11195 paralleled the level of PBR expression. PK11195 induced dephosphorylation of protein kinase B/Akt and Bad and a downregulation of Bcl-2. Collapse of the mitochondrial membrane potential preceeded PBR-ligand-induced apoptosis. No apoptosis was induced by PK11195 in parenchymal cells, despite the presence of PBR, and PK11195 had no effect in these cells on Bad phosphorylation and Bcl-2 expression. CONCLUSIONS: Transformation of HSC leads to a transient expression of PBR and renders the cells sensitive to PBR-ligand-induced apoptosis, involving protein kinase B/Akt and Bad-dependent mechanisms.

The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized madin-darby canine kidney cells.

The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain.

The inhibition of interleukin-6-dependent STAT activation by mitogen-activated protein kinases depends on tyrosine 759 in the cytoplasmic tail of glycoprotein 130.

Mitogen-activated protein (MAP) kinases stimulated by phorbol 13-myristate 12-acetate (PMA) have been shown to inhibit interleukin-6-induced activation of STAT3 (Sengupta, T. K., Talbot, E. S., Scherle, P. A., and Ivashkiv, L. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11107-11112). In the present study we demonstrate that in addition to STAT3, also tyrosine phosphorylation of STAT1, signal transducer gp130, and phosphotyrosine-phosphatase SHP2 underlies negative regulation by MAP kinases. Stimulation of Erks by basic fibroblast growth factor or a constitutively active mutant of Raf also led to down-regulation of STAT activity. Using chimeric receptor mutants we show that tyrosine 759 of glycoprotein 130 is crucial for the inhibitory effect of MAP kinases. Inhibition is also dependent on gene transcription and translation indicating that newly synthesized proteins are involved. Both PMA and basic fibroblast growth factor rapidly stimulate mRNA expression of the suppressor of cytokine signaling-3 (SOCS-3) and this induction is strongly reduced by an inhibitor of MAP kinase activation. Together with recent results demonstrating that SOCS-3 can bind in vitro to a phosphorylated tyrosine 759 peptide of glycoprotein 130 these data suggest SOCS-3 to be instrumental in the inhibition of the Janus kinase/STAT pathway by MAP kinases.

Expression of glutamine synthetase in macrophages.

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.

LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages.

Recent findings indicate that cytokine signaling can be modulated by other mediators of simultaneously activated signal transduction pathways. In this study we show that LPS and TNFalpha are potent inhibitors of IL-6-mediated STAT3 activation in human monocyte derived macrophages, rat liver macrophages and RAW 264.7 mouse macrophages but not in human hepatoma cells (HepG2) or in rat hepatocytes. Accordingly, LPS and TNFalpha were found to induce the expression of SOCS3 mRNA in each of the investigated type of macrophages but not in HepG2 cells. Using a specific inhibitor, evidence is presented that the p38 MAP kinase might be involved, especially for the inhibitory effect of TNFalpha.

The mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity.

Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor kappaB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6.

Involvement of CD95 (Apo-1/Fas) ligand expressed by rat Kupffer cells in hepatic immunoregulation.

BACKGROUND & AIMS: CD95 (Apo-1/Fas) ligand suppresses inflammatory responses in immune-privileged organs. In this study, modulation of the hepatic CD95 receptor/ligand system by interferon gamma and cyclosporin A was investigated. METHODS: CD95 receptor and ligand expression were measured at the messenger RNA level by using quantitative reverse-transcription polymerase chain reaction and immunocytochemistry in primary cultures of rat Kupffer cells, hepatocytes, and T lymphocytes. Soluble CD95 in culture supernatants was detected by enzyme-linked immunosorbent assay and apoptosis by the TUNEL method. RESULTS: Interferon gamma treatment led to an increase in CD95 ligand messenger RNA levels in Kupffer cells followed by an overexpression of the soluble CD95 receptor. Supernatants derived from 24-hour but not from 48-hour interferon gamma-treated Kupffer cells killed lymphocytes by a CD95-dependent mechanism. Cyclosporin A inhibited CD95 ligand expression in Kupffer cells and lymphocyte killing. In liver parenchymal cells, interferon gamma increased messenger RNA levels of the transmembrane CD95 isoform and sensitivity of these cells toward CD95-mediated apoptosis. CONCLUSIONS: The expression pattern of CD95 receptor and ligand in response to interferon gamma points to a coordinated interplay between Kupffer cells, hepatocytes, and T lymphocytes in which Kupffer cells may regulate programmed cell death of T lymphocytes and hepatocytes.

Organic osmolyte transport in quiescent and activated rat hepatic stellate cells (Ito cells).

Activation of hepatic stellate cells (HSCs) results in multiple alterations of cell function, but nothing is known about organic osmolytes in these cells. Organic osmolyte transport and transporter messenger RNA (mRNA) expression was studied in quiescent rat HSCs and after their transformation into alpha1-smooth muscle actin-positive myofibroblastlike cells. Quiescent stellate cells expressed in an osmosensitive manner the mRNA levels of the transporters for taurine (TAUT) and myoinositol (SMIT), whereas that for betaine was not detectable. However, these cells showed osmosensitive uptake not only of taurine and myoinositol but also of betaine. Osmosensitive betaine uptake was mediated by amino acid transport system A. After transformation into myofibroblasts, taurine and myoinositol uptake increased 5.5-fold and 4.5-fold, respectively, together with the respective transporter mRNA levels. Betaine uptake increased twofold because of osmosensitive induction of BGT1 expression. In both quiescent and activated HSCs, hypoosmotic cell swelling induced a rapid and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid-sensitive osmolyte efflux. In quiescent HSCs, hyperosmotic exposure increased the messenger RNA (mRNA) level of cyclooxygenase-2, which was counteracted by taurine but not by betaine or myoinositol. The study identifies taurine, myoinositol, and betaine as osmolytes in HSCs. Transformation of HSCs is accompanied by enhanced osmolyte transport activity and induction of the BGT1 transporter, which may be another activation marker of HSCs.

Release of osmolytes from perfused rat liver on perivascular nerve stimulation: alpha-adrenergic control of osmolyte efflux from parenchymal and nonparenchymal liver cells.

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine-sensitive increase of cell hydration by 6.5% +/- 1.2% (n = 3) and a transient phentolamine-sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2alpha, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (50 micromol/L). Single-cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and-consecutively-osmolyte content are modulated by hepatic nerves via an alpha-adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve-dependent alpha-adrenergic activation, whereas 3) KC and SEC probably do not express alpha-adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1.

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.

De novo expression of glutamine synthetase during transformation of hepatic stellate cells into myofibroblast-like cells.

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of alpha-smooth-muscle actin. In parallel with the accumulation of alpha-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13+/-0.01 micromol/h per mg of cell protein within 7-14 days. This value compares with GS activity in liver parenchymal cells of 0.57+/-0.03 micromol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.